Vaccination with recombinant neuraminidase protects against influenza virus infection in mice

While the efficacy of most influenza virus vaccines is measured by the ability to induce antibodies against the hemagglutinin (HA), antibodies against the viral neuraminidase (NA) are also correlated with less severe disease in humans and animal models. Yet, neither the amount nor the enzymatic activity of NA is standardized in current seasonal vaccines, and the breadth of NA-based protection is unknown. In the present study, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to homologous, heterologous, or heterosubtypic virus challenge. Additionally, pre- and post-vaccination human serum samples from vaccinees that received TIV were studied to compare induction of antibodies against the HA and NA. Finally, the amounts of NA in 4 different vaccine formulations from 2013-2014 were quantified using ELISA. Mice immunized with N2 were 100% protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Mice immunized with B/Yamagata/16/88 NA were 100% protected from morbidity and mortality when lethally challenged with a recent Victoria lineage strain. In our human cohorts, the increase in endpoint titers against N1 NA post-vaccination was less robust than that against HA and, as our quantification data suggests, the N1 NA amounts in seasonal vaccine formulations is quite variable. To confirm the broad protective effects of anti-influenza B NA antibodies on a monoclonal level, a panel of mouse monoclonal antibodies was generated against influenza B virus NA; several of these displayed broad reactivity in ELISA to whole virus and recombinant NA and protected against lethal influenza B virus challenge in mice when delivered at a dose of 5 mg/kg prophylactically, or therapeutically, 48 hours post-infection. Analysis of the protective epitopes is currently in progress. The demonstrated protective capacity of anti-NA antibodies suggests that targeting the NA through vaccination may offer increased protection against influenza virus infection

Immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase 1 clinical trial.

BackgroundInfluenza viruses cause substantial annual morbidity and mortality globally. Current vaccines protect against influenza only when well matched to the circulating strains. However, antigenic drift can cause considerable mismatches between vaccine and circulating strains, substantially reducing vaccine effectiveness. Moreover, current seasonal vaccines are ineffective against pandemic influenza, and production of a vaccine matched to a newly emerging virus strain takes months. Therefore, there is an unmet medical need for a broadly protective influenza virus vaccine. We aimed to test the ability of chimeric H1 haemagglutinin-based universal influenza virus vaccine candidates to induce broadly cross-reactive antibodies targeting the stalk domain of group 1 haemagglutinin-expressing influenza viruses.MethodsWe did a randomised, observer-blinded, phase 1 study in healthy adults in two centres in the USA. Participants were randomly assigned to one of three prime-boost, chimeric haemagglutinin-based vaccine regimens or one of two placebo groups. The vaccine regimens included a chimeric H8/1, intranasal, live-attenuated vaccine on day 1 followed by a non-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine on day 85; the same regimen but with the inactivated vaccine being adjuvanted with AS03; and an AS03-adjuvanted, chimeric H8/1, intramuscular, inactivated vaccine followed by an AS03-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine. In this planned interim analysis, the primary endpoints of reactogenicity and safety were assessed by blinded study group. We also assessed anti-H1 haemagglutinin stalk, anti-H2, anti-H9, and anti-H18 IgG antibody titres and plasmablast and memory B-cell responses in peripheral blood. This trial is registered with ClinicalTrials.gov, number NCT03300050.FindingsBetween Oct 10, 2017, and Nov 27, 2017, 65 participants were enrolled and randomly assigned. The adjuvanted inactivated vaccine, but not the live-attenuated vaccine, induced a substantial serum IgG antibody response after the prime immunisation, with a seven times increase in anti-H1 stalk antibody titres on day 29. After boost immunisation, all vaccine regimens induced detectable anti-H1 stalk antibody (2·2-5·6 times induction over baseline), cross-reactive serum IgG antibody, and peripheral blood plasmablast responses. An unsolicited adverse event was reported for 29 (48%) of 61 participants. Solicited local adverse events were reported in 12 (48%) of 25 participants following prime vaccination with intramuscular study product or placebo, in 12 (33%) of 36 after prime immunisation with intranasal study product or placebo, and in 18 (32%) of 56 following booster doses of study product or placebo. Solicited systemic adverse events were reported in 14 (56%) of 25 after prime immunisation with intramuscular study product or placebo, in 22 (61%) of 36 after immunisation with intranasal study product or placebo, and in 21 (38%) of 56 after booster doses of study product or placebo. Disaggregated safety data were not available at the time of this interim analysis.InterpretationThe tested chimeric haemagglutinin-based, universal influenza virus vaccine regimens elicited cross-reactive serum IgG antibodies that targeted the conserved haemagglutinin stalk domain. This is the first proof-of-principle study to show that high anti-stalk titres can be induced by a rationally designed vaccine in humans and opens up avenues for further development of universal influenza virus vaccines. On the basis of the blinded study group, the vaccine regimens were tolerable and no safety concerns were observed.FundingBill & Melinda Gates Foundation

The Influenza Virus Neuraminidase as a Vaccine Antigen and the Potential of Neuraminidase Antibodies to Protect Against Infection

The influenza virus continues to cause significant morbidity and mortality in humans, resulting in up to 50,000 deaths per year in the United States. Annual vaccination remains the recommended prophylaxis for influenza. However, vaccines must be reformulated to account for antigenic drift and, even when vaccines contain strains that antigenically match circulating strains, they display suboptimal efficacies. Two glycoproteins coat the surface of the influenza virus – the more abundant and immunodominant hemagglutinin (HA), which serves as the receptor-binding protein, and the neuraminidase (NA), an enzyme that functions to free budding viruses from infected cells. Current influenza virus vaccine strategies aim to elicit neutralizing antibodies against the HA, but past studies have demonstrated that neuraminidase inhibition titers are correlated with reduced illness and viral shedding in humans. Despite the accumulated evidence that an anti-NA immune response is beneficial, the NA content in vaccines is not standardized. Here, the potential breadth of protection afforded by NA antibodies was investigated by studying the use of NA as a vaccine antigen and by characterizing broadly cross-reactive murine monoclonal antibodies against the NA. Using baculovirus-expressed, purified protein, it was demonstrated that vaccination with adjuvanted NA was sufficient to induce protection against lethal influenza virus challenge in mice. In the same study, the N1 NA content of inactivated influenza virus vaccines from different companies was found to be highly variable. Furthermore, in humans vaccinated with standard inactivated influenza virus vaccine, the induction of serum NA titers was significantly lower than that of HA titers. In the second part of this dissertation, panels of monoclonal antibodies were generated against the N8 NA of an emerging H10N8 influenza virus strain and against the NA of influenza B virus. Monoclonal antibodies against the influenza B virus NA displayed in vivo prophylactic and therapeutic protection in mice, robustly activated antibody-dependent cellular cytotoxicity (ADCC) in vitro, and displayed neuraminidase inhibition against an oseltamivir-resistant influenza B virus. As a whole, our data strongly suggest that targeting the influenza virus NA may be beneficial when designing novel influenza virus vaccines or antibody-based therapeutics

Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice

In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the NA content in 4 different inactivated-virus vaccine formulations from the 2013-2014 season via Western blot assay and enzyme-linked immunosorbent assay quantification, we found that the amount of NA does indeed vary across vaccine brands. We also measured hemagglutinin (HA) and NA endpoint titers in pre- and postvaccination human serum samples from individuals who received a trivalent inactivated seasonal influenza vaccine from the 2004-2005 season; the induction of NA titers was statistically less pronounced than the induction of HA titers. The demonstrated homologous and heterologous protective capacity of recombinant NA suggests that supplementing vaccine formulations with a standard amount of NA may offer increased protection against influenza virus infection

An immuno-assay to quantify influenza virus hemagglutinin with correctly folded stalk domains in vaccine preparations.

The standard method to quantify the hemagglutinin content of influenza virus vaccines is the single radial immunodiffusion assay. This assay primarily relies on polyclonal antibodies against the head domain of the influenza virus hemagglutinin, which is the main target antigen of influenza virus vaccines. Novel influenza virus vaccine candidates that redirect the immune response towards the evolutionary more conserved hemagglutinin stalk, including chimeric hemagglutinin and headless hemagglutinin constructs, are highly dependent on the structural integrity of the protein to present conformational epitopes for neutralizing antibodies. In this study, we describe a novel enzyme-linked immunosorbent assay that allows quantifying the amount of hemagglutinin with correctly folded stalk domains and which could be further developed into a potency assay for stalk-based influenza virus vaccines

A cross-clade H5N1 influenza A virus neutralizing monoclonal antibody binds to a novel epitope within the vestigial esterase domain of hemagglutinin

10.1016/j.antiviral.2017.06.012Antiviral Research144299-31

Development of capture ELISA to measure correctly folded HA stalk domain.

<p>(<b>A</b>) and (<b>B</b>) describe the underlying concept of the assay. The assay was primarily developed to detect HA with conformationally intact stalk epitopes. The capture mAb in this assay recognizes the HA head linear epitope. On the other hand, the detection mAb recognizes the conformational epitope in the HA stalk. <b>(A)</b> If the stalk of the HA is properly folded, the detection mAb can bind and thus produces a signal. <b>(B)</b> If the stalk is misfolded, no signal is observed since the detection mAb cannot recognize the HA stalk. <b>(C)</b> HA concentration was calculated using a curve-fit model similar to parallel line analysis in which EC₅₀ values are used to determine relative amounts. An EC₅₀ analysis is shown using a recombinant protein standard with a known concentration for reference. The shift in EC₅₀ between protein standard and test samples was then used to calculate the HA content of test samples. <b>(D)</b> The test samples were treated with 0.05% zwittergent for 1h to solubilize the influenza virus membrane and prevent transmembrane-driven rosette formation. EC₅₀ values of virus preparations treated with or without zwittergent were calculated. Bars represent mean with error bars representing standard error of the mean.</p

Viruses tested for stability during storage at 4°C and 27°C.

<p>Viruses tested for stability during storage at 4°C and 27°C.</p